Project Summary/Abstract Transient receptor potential (Trp) channels are involved in many fundamental cell functions and associated with many disease states (65). Trpm channels are a subgroup of Trp channel superfamily. Trpm2 is expressed in many tissues including heart, vasculature, hematopoietic cells and brain. Trpm2 is activated by ADP-ribose (ADPR) and H2O2 and mediates Ca2+ influx into the cell. Trpm2 has an essential role in susceptibility to oxidative stress. The existing paradigm is that activation of Trpm2 induces cell death by sustained increases in [Ca2+]i, or mediates enhanced chemokine production in hematopoietic cells thereby aggravating inflammatory response and tissue injury. Quite unexpectedly, our recent data demonstrated that Trpm2 is essential in cellular bioenergetics maintenance in both the heart and neuroblastoma; and that Trpm2 actually protects the heart and neuroblastoma from oxidative-stress induced injury. Trpm2 is overexpressed in many cancers and the level of Trpm2 overexpression correlated with decreased patient survival and increased propensity for metastasis in some tumors. Based on these observations, Trpm2 has increasingly become a rational target for cancer therapy. In support of this concept, we showed that neuroblastoma xenografts over- expressing the dominant-negative Trpm2-S had significantly suppressed growth and enhanced sensitivity to doxorubicin (Doxo). In addition, targeting Trpm2 was recently shown to promote cell death in T cell leukemia. Therefore, although Trpm2 inhibition can enhance the therapeutic effect of chemotherapy (e.g., Doxo), it may inadvertently disturb mitochondrial energy metabolism and redox balance and as such, aggravate existing ischemic heart disease and Doxo-induced cardiomyopathy. The study of mechanisms by which Trpm2 protects the heart is thus timely and will significantly contribute to the nascent field of onco-cardiology. We are the first to demonstrate that Trpm2 channels are expressed at the sarcolemma and t-tubules and measure Trpm2 channel activity in adult mouse LV myocytes. We have obtained preliminary data indicating Trpm2 activation phosphorylates proline-rich tyrosine kinase 2 (Pyk2), one of Ca2+- and redox- sensitive non-receptor tyrosine kinase in the heart, that subsequently translocates to mitochondrial matrix. We have shown that phosphorylated Pyk2 (pPyk2) can enhance mitochondrial Ca2+ uptake through mitochondrial Ca2+ uniporter (MCU). To explore the function of Trpm2 in cardiac myocytes, we have generated both global Trpm2 knockout (gKO) and cardiac-specific Trpm2 KO (cKO) mice. Compared to WT myocytes, gKO myocytes had decreased expression of proteins involved in mitochondrial function and oxidative defense. Functionally, gKO myocytes had lower mitochondrial membrane potential (??m), reduced MCU activity, decreased mitochondrial Ca2+ uptake, increased mitochondrial superoxide (O2-.) levels, decreased oxygen consumption rate (OCR) and lower ATP levels: indicating compromised cellular bioenergetics and redox balance. Although baseline cardiac performance was similar between WT and gKO hearts, the deleterious effects of abnormal cellular energetics and redox balance were manifest in 2 models of oxidative stress: Doxo cardiomyopathy and ischemia/reperfusion (I/R) injury. Taken together, we hypothesize that tonic activation of Trpm2 by basal levels of H2O2 emitted by respiring cardiac mitochondria under physiological conditions facilitates the essential mitochondrial Ca2+ uptake through pPyk2-mediated MCU activation for bioenergetics maintenance and redox balance. Under stress, ablation or inhibition of Trpm2 results in insufficient cellular bioenergetics and augmented oxidative stress, causing compromised myocardial performance. Consequently, targeting Trpm2 channels in cancer cells without adequate cardiac protection may have serious untoward cardiac side effects including increased mortality. We propose the following to evaluate Trpm2 function in the heart in health and disease: Specific Aim 1: What is the signaling mechanism for Trpm2-mediated increases in cellular energy production? We hypothesize that Trpm2 activation phosphorylates Pyk2 and the activated pPyk2 translocates to mitochondrial matrix, which leads to enhanced mitochondrial Ca2+ uptake through MCU, thereby enhancing Ca2+ -regulated ATP generation. Specific Aim 2: What is the mechanism for Trpm2-mediated decreases in ROS generation? We hypothesize that Trpm2-mediated Ca2+ influx promotes mitochondrial electron transport activity thereby reducing mitochondrial O2-. generation, rather than by modulating NADPH oxidase (NOX) activity to decrease ROS. Trpm2 ablation also results in decreased anti-oxidant protein profiles. Specific Aim 3: Is Trpm2-mediated Ca2+ influx necessary for improvement in in vivo cardiac function post-I/R or Doxo treatment of cKO hearts? We will modulate Trpm2-mediated Ca2+ entry in cardiac myocytes by expressing WT Trpm2, loss-of-function (E960D), increased Ca2+ selectivity (Q981E/P983Y), or inactivation (P1018L) Trpm2 mutants in cKO hearts, subjected to either I/R or Doxo treatment and cardiac performance evaluated. We will also manipulate Pyk2 activity in cKO hearts and assess the effects on cardiac function post-I/R or Doxo treatment. The effects of Trpm2 mutants on expression of proteins involved in oxidative defense will also be evaluated.